1. Field of the Invention
The present invention generally relates to the art of biotechnology, and more specifically to a method including scanning a gene probe array to produce data having a dynamic range which exceeds that of the scanner.
2. Description of the Related Art
U.S. Pat. No. 5,143,854, entitled "LARGE SCALE PHOTOLITHOGRAPHIC SOLID PHASE SYNTHESIS OF POLYPEPTIDES AND RECEPTOR BINDING SCREENING THEREOF", issued Sep. 1, 1992 to Michael Pirrung et al, discloses a basic method for synthesizing polypeptide arrays on a substrate by attaching photoremovable groups to the surface of the substrate, exposing selected regions of the substrate to light to activate those regions, attaching an amino acid monomer with a photoremovable group to the activated regions, and repeating the steps of activation and attachment until polypeptides of the desired length and sequence are synthesized. The subject matter of this patent is incorporated herein by reference in its entirety.
The polypeptides of the array are known as "probes" and act as receptors. The probe array is "hybridized" by exposure to a polymer substance which is to be analyzed. The polymer acts as a ligand or target and will bond to one or more probes which have a complementary sequence of bases. The polymer is tagged with a fluorescent reporter group or marker which emits light upon radiation with light of a suitable wavelength. The array is optically scanned by sequentially irradiating the probes in a rectangular pattern and sensing the emitted light intensity at each probe location.
The fluorescent intensity increases with the bonding strength of a ligand to a receptor. Since the probe sequence at each location is known, the unknown polymer ligand can be identified as being complementary to the probe which produces the greatest value of fluorescent intensity.
Improved methods for scanning a probe array are disclosed, for example, in U.S. Pat. No. 5,795,716, entitled "COMPUTER-AIDED VISUALIZATION AND ANALYSIS SYSTEM FOR SEQUENCE EVALUATION", issued Aug. 18, 1998 to Mark Chee et al. The subject matter of this patent is incorporated herein by reference in its entirety.
The methods disclosed in the above referenced patents have been yet further developed to produce a genetic analysis system based on the GeneChip.RTM. Probe Array, a product of Affymetrix.RTM. of Santa Clara, Calif. The GeneChip includes a large number of polymer probes which are selected to be complementary to an unknown genetic or other material which is to be analyzed. The probes are formed on a substrate in the manner described above, and generally differ from each other by one base. The probe array further includes a protective casing which protects the substrate and attached probes during storage and handling.
The Affymetrix system further includes a fluidics station in which the probe is hybridized by exposure to the genetic or other substance which is to be analyzed. The hybridized array is then tagged with a fluorescent marker and scanned in an optical scanner, and the light intensity at each point is sensed and stored in a digital memory. The data is then processed at a computer workstation to determine the identity of the unknown substance.
The Affymetrix Gene Chip system can be advantageously utilized in a number of biotechnological areas. One of these areas is gene expression analysis. Differential expression data can provide a clear understanding of cellular pathways and identify valuable candidates for drug discovery. GeneChip expression probe arrays simplify genomic research by quantitatively and simultaneously monitoring the expression of thousands of genes.
GeneChip expression probe arrays are capable of identifying mRNA expression level changes of greater than twofold between experiments and are able to detect mRNA transcripts from the level of only a few copies per cell to more than several hundred thousand copies per cell.
In contrast to prior art spotting methods in which a single clone is used to analyze each mRNA, GeneChip expression arrays use approximately 20 pairs of specific oligonucleotide probes to interrogate each transcript. This probe pairing strategy helps identify and minimize the effects of non-specific hybridization and background signal to enable sensitive and accurate recognition of low-intensity hybridization patterns from mRNA. This makes it possible to specifically detect individual gene transcripts and splice variants and differentiate among closely related members of gene families.
The GeneChip expression arrays contain probes corresponding to a number of reference and control genes. These reference standards make it possible to normalize data from different experiments and compare multiple experiments on a quantitative level.
Another advantageous application of the Affymetrix GeneChip system is polymorphism analysis. This enables researchers to identify and map the thousands of genes comprising the human genome and to identify the base present at specific sequence locations. After the sequences are determined for the first time, it becomes increasingly valuable to identify polymorphisms (or variations) in these genes and to understand how these polymorphisms impact biological function and disease. These association studies require the analysis of DNA samples from a large number of affected and unaffected individuals for each disease under study.
The GeneChip SNP (Single Nucleotide Polymorphism) mapping assays accelerate genetic analysis by minimizing labor, data analysis time and total time required to run complex genotyping studies. The mapping assays enable study of the links between polymorphisms and disease, the mechanisms that lead to disease, and patient response to treatment.
Yet another area in which the Affymetrix GeneChip technology can be advantageously applied is disease management. Researchers are beginning to unravel the mysteries of how genetics impacts human health. The GeneChip technology enables the rapid and accurate analysis of relevant genetic information and has the potential to transform diagnosis into a high-value disease management paradigm.
Disease management is an emerging field focussed on improving the effectiveness of healthcare by using genetic information to improve and guide therapy. Gene expression profiles and polymorphisms that correlate with a specific disease or therapeutic response have the potential to become critical information for disease management.
GeneChip arrays are advantageously applicable in the study of more effective patient management in the areas of infectious disease, cancer and drug metabolism. This enables researchers to understand the genetic basis and progression of disease and patient response to treatment. GeneChip assays are being used to correlate specific mutations with patient outcomes under varied therapeutic drug regimes.
With data gathered through these studies, scientists can develop more detailed prognoses, drug therapies and treatment strategies. Affymetrix has developed the GeneChip CYP450 assay which is the first tool that enables efficient and simultaneous analysis of multiple genotypes associated with drug metabolism defects. These defects can make even innocuous drugs dangerous for certain persons, keeping some potentially valuable therapeutics off the market.
Although the biotechnological applications described above constitute major areas in which the GeneChip system is a desirable analytic tool, these applications are presented as being exemplary only, and not limitatative of the scope of the present invention. On the contrary, the invention as described herein is applicable to numerous and varied arts in which unknown substances are to be identified and, as will be described in detail below, a widely varied range of technologies in which data is to be provided which has a dynamic range larger than a sensor used to obtain the data.
As described briefly above, a hybridized gene probe array is optically scanned by irradiating the individual probes with light of a certain wavelength, and sensing the light intensity resulting from fluorescence of the probes. The ligand polymers of the unknown substance are tagged with fluorescent markers. The markers which are most commonly used are phycoerythrin, which produces maximum or peak fluorescence at a wavelength of 578 nm, and fluorescein, which produces peak fluorescence at a wavelength of 520 nm.
An optical scanner has been developed as a joint project of Affymetrix and the Hewlett Company of Palo Alto, Calif. The scanner is an improvement over previously used scanners and is commercially available from Hewlett Packard under the product designation "HP G2500A Gene Array Scanner". Whereas previously available scanners could only read GeneChip probe arrays with 65,000 probe cells, the Hewlett Packard scanner can read probe arrays with up to 400,000 cells and provide data on thousands of genes and mutations. The scanner focuses a laser beam onto a 3 micron section of a variable size probe array feature, and can detect emitted light at wavelengths of 570 nm and 530 nm for the two common fluorescent markers described above. Note that the "530 nm" setting is a nominal shorthand for using a filter in front of the photomultiplier that permits light of wavelength about 525 nm to 555 nm to pass. Likewise, the "570 nm" setting is a nominal shorthand indicating use of a filter in front of the photomultiplier that allows light of wavelength &gt;570 nm to pass.
In certain applications, however, even the Hewlett Packard scanner is incapable of providing useful intensity readings using conventional markers for very low levels of fluorescent intensity. For this reason, new fluorescent markers are being developed which emit more light and enable useful measurements to be made under conditions which were not previously possible. An example of such a new marker is phycoerythrin-labeled target DNA as will be described in detail below.
Although the greater level of signal obtained was a great boon, it also created a problem. In some cases, the signal intensity was so high that a saturated signal was obtained using the Hewlett Packard scanner. It was not possible to get useful readings at both the high and low ends of the intensity range. This was due to a limitation in the range of detection (dynamic range) of the photomultiplier tube in the Hewlett Packard scanner. Although it is possible to reduce the gain of the tube to prevent saturation, this may result in loss of sensitivity and accuracy at the low end of the range.
In view of the above, a specific need exists in the art for a method of scanning a GeneChip probe array which is hybridized with high intensity fluorescent markers and obtain useful data in both the high and low ranges of the intensity scale. In order to accomplish this goal, it is necessary for the method to produce data having a dynamic range which is greater than that of the scanner itself.
More generally, a need exists in the art for a method of obtaining data from a scanner which is adapted to sense any of a number of varied parameters, and to similarly produce data having a dynamic range which is greater than that of the scanner.